The immunogenic properties of plasmid DNA and recombinant adenovirus (Ad) encoding the carcinoembryonic antigen (CEA) were examined in mice by measuring both the amplitude and type of immune response, and the immunogenicity of codon usage optimized cDNA encoding CEA (CEAopt) was assessed both in C57Bl/6 and CEA transgenic mice. Vectors were injected into quadriceps muscle either alone or in combination, and plasmid DNA was electroporated to enhance gene expression efficiency and immunogenicity. Injection of plasmid pVIJ/CEA followed by Ad‐CEA boost elicited the highest amplitude of both CD4⁺ and CD8⁺ T‐cell response to the target antigen, measured by both IFNγ‐ELIspot assay and intracellular staining. Vectors carrying cDNA of CEAopt expressed a greater amount of the CEA protein than their wild‐type counterparts, and this enhanced expression was associated with greater immunogenicity. Both CD4⁺ and CD8⁺ T‐cell epitopes were mapped in the C‐terminal portion of the protein. In CEA transgenic mice, only immunization based on repeated injections of pVIJ/CEAopt followed by Ad‐CEAopt was able to elicit a CEA‐specific CD8⁺ T‐cell response, whereas the wild‐type vectors did not break tolerance to this target antigen. MC38‐CEA tumor cells injected s.c. in CEA transgenic mice vaccinated with CEAopt vectors exhibited delayed growth kinetics. These studies demonstrate that this type of genetic vaccine is highly immunogenic and can break tolerance to CEA tumor antigen in CEA transgenic mice. © 2005 Wiley‐Liss, Inc.